Compositions and methods for modulating AT2R activity

ABSTRACT

New polypeptide agonists of AT2R are disclosed, as well as pharmaceutical compositions comprising the agonists, methods of their use in the treatment of diseases, conditions or disorders characterized by insufficient AT2R activity or excessive AT1R activity, and methods of their use as laboratory reagents for research purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/US15/061597, entitled “Compositions and Methods for Modulating AT2R Activity”, and filed on Nov. 19, 2015, which claims priority to U.S. Provisional application 62/081,839, entitled “Compositions and Methods for Modulating AT2R Activity”, and filed on Nov. 19, 2014, both of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 19, 2016, is named 220935200122WO1_SL.txt and is 6,228 bytes in size.

FIELD OF THE INVENTION

The invention relates to the fields of molecular biology and biochemistry, and in particular to the development of pharmaceuticals for the modulation of the activity of the Angiotensin II Type 2 Receptor (AT2R).

BACKGROUND

The Angiotensin II Type 2 Receptor AT2R is a member of the anti-inflammatory/vasodilative branch of the renin-angiotensin system (RAS). AT2R-activation ameliorates cardiovascular diseases and stroke, attenuates cancers and exerts a neuroprotective role (1-17).

AT2R is a transmembrane receptor protein comprising a sequence of 363 amino acids which form seven-transmembrane domains. The three-dimensional structure of AT2R has not yet been resolved, but it contains five potential glycosylation sites and a conserved lysine residue (Lys 199 or K199) that is critical for ligand-protein interaction. AT2R also contains a potential protein kinase C phosphorylation site in the second intracellular loop (18).

AT2R belongs to the G-Protein Coupled Receptor (GPCR) family of proteins. AT2R activation stimulates various protein phosphatases (e.g., SHP1, MKP1 and PP2A) and inhibits cancer cell growth. AT2R-mediated activation of the bradykinin/nitric oxide/cGMP pathway and the prostaglandin I2-IP receptor pathway contribute to its vasodilatory effects (19-23). However, many of the signaling mechanisms activated by the AT2R are G-protein independent and involve direct interactions between AT2R and other cellular proteins. AT2R interacts with a family of AT2R-interacting proteins (ATIPs) involved in neuronal differentiation, vascular remodeling and tumor suppression via its C-terminal cytoplasmic domain (CCD) (24). It has been shown that the AT2R interacts with the ErbB family receptors and Na⁺/H⁺ exchanger NHE6 via its third intracellular loop (ICL) and the CCD, and that the third ICL of the AT2R is involved in attenuating Angiotensin II Type 1 Receptor (AT1R) signaling (25, 26). AT2R-mediated apoptosis also requires the third ICL (27, 28). Interestingly, deletion of the CCD reduces affinity of AT2R to Angiotensin II (Ang II), but increases its affinity to the peptide ligand CGP42112A and enhances Ang II-induced cGMP reduction (29, 30). These observations highlight the roles of the third ICL and the CCD in AT2R signaling.

AT2R down-regulation is seen in Parkinson's Disease (PD) (31). Although not much is known about the role of AT2R in PD, it is known that AT2R activation causes differentiation of dopaminergic neurons from mesencephalic precursors (32). Additionally, AT2R activation is neuroprotective to cultured mid-brain dopaminergic neurons, whereas use of an AT2R antagonist eliminates the neuroprotective effects (33).

Early studies from Mendelsohn et al. (1988) and Unger et al. (1988) established, using biochemical and pharmacological approaches, the existence of a renin-angiotensin system in the brain (34, 35). The various components (e.g., angiotensin-converting enzyme (ACE), Ang II and Ang II receptors) are found in areas of the brain involved in the regulation of fluid and electrolyte balance and in the regulation of arterial pressure (36, 37), as well as in structures involved in cognition, behavior and locomotion. Interestingly, all of these components, and in particular AT2R, are highly expressed during fetal life. This suggests that they could play important roles during development. As reported by Nuyt et al. based on studies conducted in fetal and neonatal rats, AT2R mRNA appeared early (e.g., as early as embryonic day 13) in the differentiating lateral hypothalamic area, but transiently in various developing/differentiating brain structures (38). In most areas, the ontogeny of AT2R mRNA expression is highly correlated with the maturation and differentiation of the different areas themselves (as in the cerebellum, inferior olivary complex, and medullary motor nuclei innervating the tongue, perioral, and jaw muscles, where AT2R expression dramatically diminished in the mature neurons).

From studies conducted in cell lines, it appears that activation of AT2R during development is involved in neurite elongation, neuron migration, neuronal death and survival balance, as well as in the establishment and maintenance of synaptic connections. In the adult rat, AT2R was found at high levels in the medulla oblongata (which controls autonomous functions), in septum and amygdala (which are associated with anxiety-like behavior), in the thalamus (which is associated with sensory perception), in the superior colliculus (which controls eye movements in response to visual information and is linked to blink hyperexcitability in Parkinson's), as well as in the subthalamic nucleus and in the cerebellum (areas associated with learning of motor functions) (39, 40, 41).

According to Bottari et al., AT2Rs are found on neurons, but not on astrocytes or glial cells. The presence of AT2R in restricted brain areas of the adult and its wide distribution in the fetus (in many differentiating structures and nuclei) are indicative of a role in neuronal function and neuronal development respectively (42). Accordingly, using cells of neuronal origin and models of neuronal regeneration, AT2R was found to be involved in the regulation of apoptosis and cell differentiation. Apart from its transient expression in many structures during development, expression of AT2R increases in the brain after cellular damage, which shows that it plays a role in neuronal wound healing. In addition to neuronal differentiation, which is of paramount importance in nerve regeneration, AT2R also stimulates differentiation of hematopoietic cells, a key process during regeneration and reconstruction (31).

Ischemic damage is characterized by infiltration of a number of hematopoietic cells such as platelets, macrophages, and leukocytes (43). Significantly, AT2R has the capacity to induce differentiation of human monocytes into dendritic cells (44), indicating a potential protective effect. Confirming this protective effect of AT2R is the observation that ischemic damage was found to be greater in mice with hematopoietic cells deleted in AT2R expression (45). These findings show that expression and activation of AT2R in hematopoietic cells is part of its beneficial effect following brain injury (46).

Renal dopamine D₁-like receptors (D₁Rs) and AT2Rs are important natriuretic receptors counter-balancing AT1R-mediated tubular sodium reabsorption. In uninephrectomized, sodium-loaded Sprague-Dawley rats, direct renal interstitial infusion of the highly selective D₁R agonist fenoldopam induced a natriuretic response that was abolished by the AT2R-specific antagonist PD-123319 or by the microtubule polymerization inhibitor nocodazole but not by the actin polymerization inhibitor cytochalasin D. The results demonstrate that D₁R-induced natriuresis requires AT2R recruitment to the apical plasma membranes of renal proximal tubule cells in a microtubule-dependent manner involving an adenylyl cyclase/cAMP signaling pathway. These studies provide novel insights regarding the mechanisms whereby renal D₁Rs and AT2Rs act in concert to promote sodium excretion in vivo (47).

Treatments of primary neurons with Compound 21 (C21), an AT2R agonist, improved functional recovery in experimental spinal cord injury through promotion of axonal plasticity and through neuroprotective and anti-apoptotic mechanisms (48).

Even though AT2R belongs to the GPCR family of proteins, its signaling mechanisms are atypical and remain elusive. Activated AT2R induces a vasodilator cascade of bradykinin (BK)/Nitric Oxide/cGMP, stimulates various protein phosphatases (e.g., SHP1, MKP1 and PP2A) and inhibits cancer cell growth (19-23). AT2R also interacts with a family of AT2 receptor interacting proteins (ATIPs) involved in neuronal differentiation, vascular remodeling and tumor suppression via its CCD (49, 50). Chronic AT1 Receptor blocker (ARB) treatment can result in redirecting Ang II to AT2R that is usually co-expressed with AT1R in cardiovascular tissues, leading to increased AT2R activation, and enhanced AT1R-AT2R cross-talk. In AT2R knock-out mice, ARBs failed to attenuate acute-phase post-infarction remodeling indicating that AT2R is required for the cardioprotective effects of ARBs (17).

Cardiovascular protective effects of AT2R are highlighted by the fact that moderate cardiac-specific AT2R overexpression protects the heart from ischemic injury (16).

The inflammatory cascade contributing to the development of cardiovascular disease (CVD) has been rapidly elucidated over the past decade, inspired by the marked increase in disease prevalence. To put this in perspective, nearly 70% of all Type 1 Diabetes Mellitus (T1DM) fatalities are attributed to the condition (51). Increased activation of the pro-inflammatory AT1R is seen in cardiovascular disease and hypertension (3). In general, increased AT1R activation up-regulates pro-inflammatory and pro-cancerous proteins such as nf-kb, IL-6 (52, 53, 54).

Diabetic nephropathy is marked by increased basal levels of certain cytokines (e.g., TNF-alpha, IL-6) and therefore experimental treatments have focused on modulating these same markers. Multiple studies have revealed that levels of cytokines in serum and urine are positively correlated with the progression of the disease. Particularly related to the pathogenesis of nephropathy, molecules such as IL-6 have been identified as being responsible for altering the permeability of vascular endothelial cells and the development of basement membrane thickening, respectively (55).

Chronic activation of RAS systemically and locally elevates Ang II. Ang II then binds to AT1R and induces signaling pathways that promote muscle constriction, salt and water retention, fibrosis, hypertrophy and hyperplasia that underlie many of the metabolic diseases and poor cardiovascular and renal prognosis. Blockade of RAS can be exerted at multiple levels: via inhibition of Renin, ACE, or AT1R signaling (1, 2, 5, 9). Efficient RAS blockers at all these levels have been developed and are currently in use to block over-activation of RAS and to offer protection from RAS-related metabolic diseases including diabetes (2).

However, evidence from randomized clinical trials such as the Aliskiren Trial in Type 2 Diabetes Using Cardio-Renal Endpoints (ALTITUDE) and the Ongoing Telmisartan Alone and in Combination With Ramipril Global Endpoint Trial (ONTARGET) shows that dual RAS blockade was not beneficial compared to monotherapy in preventing serious outcomes in patients with known vascular disease or diabetes with end-organ damage (56-58). Clinical evidence supporting the association of RAS inactivation to renal diseases and basic research on RAS have begun to unveil the intricate self-regulatory signaling loops that fine-tune RAS activation and the adaptive/protective role of RAS in many tissues (9). In this context, Ang II manifests its vasodilative/cardiovascular protective/anti-inflammatory effects when it activates AT2R.

Mitochondria also express a local angiotensin system (MAS). Importantly, AT2R located in the inner membrane of mitochondria plays a significant role in mediating mitochondrial respiration. It is known that during the aging process, mitochondrial AT2R expression is reduced, while the expression of the pro-inflammatory AT1R is increased (62). The critical role of MAS in aging indicates that this system plays a role in Alzheimer's Disease (AD) development. In further support, it has been shown that amyloid-beta leads to the increased oligomerization and loss of function of the AT2R receptor, which is thought to contribute to pathogenesis of the disease (63,64).

AT1R blockers (ARBs) have been reported to reduce age related mitochondrial dysfunction, attenuate hypertension-induced renal mitochondrial dysfunction, and protect against cardiac mitochondrial dysfunction in the setting of acute ischemia (62,65). Inhibition of AT1R by ARBs theoretically allows more Ang II to bind and activate AT2R. Therefore, elevation of the opposing AT2R system will provide additional improvements in mitochondrial function. Disruption of AT1R was associated with an increased number of mitochondria and up-regulation of the prosurvival genes nicotinamide phosphoribosyltransferase (Nampt) and sirtuin 3 (Sirt3) in the kidney, leading to marked prolongation of life span in mice (66). Of these genes, Sirt3 is known to regulate AD-mediated stress (67).

NHE6 is a mitochondrial protein located in the inner membrane of mitochondria known to improve cognition and memory, and mutations in the NHE6 gene are linked with various neurological disorders such as autism and Christianson's Syndrome (68-70). It has been shown that AT2R interacts with NHE6 via its third ICL. AT2R inhibits AT1R-mediated threonine/tyrosine phosphorylation of NHE6 (71-73). This indicates that AT1R-mediated phosphorylation is a tag for degradation that is prevented via the AT2R-NHE6 interaction.

MCL-1 (myeloid leukemia cell differentiation protein) is a protein that is a member of the Bcl-2 family (74). There are two distinct variants of MCL-1, based on alternative splicing: a long form and two shorter isoforms. The long form (MCL-1L) contains 312 residues, while the short isoforms (MCL-1S) is 271 residues, with the 41 residue difference occurring at the C terminus. MCL-1L contains the standard domains found in the Bcl-2 family including BH1, BH2, BH3, and a transmembrane domain. In contrast, MCL-1S only contains the BH3 domain. This alternative splicing leads to two vastly different biological functions for MCL-1L and MCL-1S. Specifically, MCL-1L is known to be anti-apoptotic while the MCL-1S in complete contrast is pro-apoptotic (75,76). The BH3-like domain region of MCL-1S can bind and dimerize with MCL-1L (77). This interaction inhibits MCL-1 biological activity and therefore MCL-1S is an antagonist to MCL-1L.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, upon the identification of a class of specific small agonists which bind to the Angiotensin 2 Type II Receptor (AT2R), thereby activating the receptor. The agonists are useful as research, diagnostic and therapeutic reagents, particularly in methods for research, diagnosis and treatment relating to diseases, conditions or disorders characterized by insufficient AT2R activity or excessive AT1R activity. The AT2R agonists of the invention are polypeptides (or polypeptide derivatives or analogues) comprising 6 amino acid residues with the generic sequence A1-A2-A3-A4-A5-A6, where A1 is Lys; A2 is Pro, 3Hyp or 4Hyp; A3 is Leu or lie; A4 is Lys, A5 is Pro, 3Hyp or 4Hyp, and A6 is Trp (SEQ ID NO. 1). Thus, the AT2R. agonists of the invention include polypeptides (or polypeptide derivatives or analogues) comprising, consisting of, or consisting essentially of any of the sequences: Lys-Pro-Leu-Lys-Pro-Trp (SEQ ID NO. 2); Lys-3Hyp-Leu-Lys-Pro-Trp (SEQ ID NO. 3); Lys-4Hyp-Leu-Lys-Pro-Trp (SEQ ID NO. 4); Lys-Pro-Ile-Lys-Pro-Trp (SEQ ID NO. 5); Lys-3Hyp-Ile-Lys-Pro-Trp (SEQ ID NO. 6); Lys-4Hyp-Ile-Lys-Pro-Trp (SEQ ID NO. 7); Lys-Pro-Leu-Lys-3Hyp-Trp (SEQ ID NO. 8); Lys-3Hyp-Leu-Lys-3Hyp-Trp (SEQ ID NO. 9); Lys-4Hyp-Leu-Lys-3Hyp-Trp (SEQ ID NO. 10); Lys-Pro-Ile-Lys-3Hyp-Trp (SEQ ID NO. 11); Lys-3Hyp-Ile-Lys-3Hyp-Trp (SEQ ID NO. 12); Lys-4Hyp-Ile-Lys-3Hyp-Trp (SEQ ID NO. 13); Lys-Pro-Leu-Lys-4Hyp-Trp (SEQ ID NO. 14); Lys-3Hyp-Leu-Lys-4Hyp-Trp (SEQ ID NO. 15); Lys-4Hyp-Leu-Lys-4Hyp-Trp (SEQ ID NO. 16); Lys-Pro-Ile-Lys-4Hyp-Trp (SEQ ID NO. 17); Lys-3Hyp-Ile-Lys-4Hyp-Trp (SEQ ID NO. 18); and Lys-4Hyp-Ile-Lys-4Hyp-Trp (SEQ ID NO. 19). In some embodiments, when A2 is Pro, A5 is 3Hyp or 4 Hyp. In some embodiments, when A5 is Pro, A2 is 3Hyp or 4-Hyp.

Thus, in one aspect, the invention provides AT2R agonist compositions, including reagent grade and pharmaceutical compositions comprising an AT2R agonist, optionally in lyophilized form or in combination with a pharmaceutically acceptable carrier.

In another aspect, the invention provides methods for activation of AT2R in a mammalian cell in vitro using an AT2R agonist composition of the invention. In some embodiments, the method is used to as a control to characterize and/or quantify downstream effects of receptor activation (e.g., effects on mammalian target of rapamycin (MTOR), NHE6, ErbB3, Nitric Oxide Synthase activity, MCL-1 and prostaglandin 12-IP). The AT2R agonist reagent compositions can also be used in vivo as a control for, or competitive or non-competitive inhibitor of, activation of AT2R or inactivation of AT1R in response to treatments with other compounds or drug candidates.

In another aspect, the invention provides methods for the treatment of a subject diagnosed with, at risk of, or otherwise in need of treatment for any diseases, conditions or disorders characterized by insufficient AT2R activity or excessive AT1R activity. The methods involve the administration of a therapeutically effect amount of the AT2R agonist of the invention in a pharmaceutically acceptable carrier to a patient in need thereof. Similarly, the invention provides the AT2R agonists of the invention for use in the preparation or manufacture of a medicament for the treatment of a subject diagnosed with, at risk of, or otherwise in need of treatment for any diseases, conditions or disorders characterized by insufficient AT2R activity or excessive AT1R activity, or insufficient activity of pathways which are downstream of AT2R signaling (e.g., diseases, conditions or disorders characterized by insufficient MTOR, NHE6, ErbB3, Nitric Oxide Synthase, MCL-1 or prostaglandin 12-IP activity or production). Diseases, conditions or disorders suitable for treatment by the methods and with the medicaments of the invention include, without limitation, (a) chronic inflammation caused by over-activation of the AT1R or under-activation of the AT2R; (b) diseases, conditions or disorders for which increasing the circulating levels of at least one MCL-1 isoform is indicated, (c) AT1R-mediated hypertension, (d) AT2R-mediated hypertension and/or cardiovascular disease, (e) AT2R-mediated neurodegenerative disorders, including but not limited to Alzheimer's Disease, Parkinson's Disease, ALS, and age-related mental deterioration, (f) neural injuries (e.g., spinal cord injuries, stroke, ischemia reperfusion injury), (g) cancers, (h) pre-eclampsia, (i) diabetes complications (e.g., retinopathy, pancreatic cell death, metabolic syndrome), (j) inflammation-mediated nephropathy, (k) inflammation-mediated liver disease (e.g., liver cancer, liver failure), (l) inflammation-mediated cardiovascular disease (e.g., myocardial ischemia and injury, myocardial fibrosis, heart attack), (m) pancreatitis, (n) insufficient muscle mass (e.g., muscle wasting due to illness or cancer treatments) (o) AT1R-mediated NHE6 degradation, (p) insufficient mitochondrial activity, and (q) diseases, conditions or disorders characterized by insufficient MTOR, NHE6, ErbB3, Nitric Oxide Synthase, MCL-1 or prostaglandin 12-IP activity or production.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

FIGS. 1A and 1B show that an NP-6AK agonist increases MCL-1 expression in female mouse HL-1 cardiomyocytes (FIG. 1A) and human CAVSMCs (FIG. 1B). CGP, a partial agonist was unable to increase MCL-1. Addition of PD, an AT2R antagonist prevents NP-6AK-mediated up regulation of MCL-1, indicating that this effect is via AT2R.

FIG. 2 shows immunofluorescence staining of SH-SY5Y cells subjected to treatments (24 hours) using an NP-6AK agonist with anti-MCL-1 antibody and nuclear stain DAPI. n≥80 and *p<0.01 compared to control. The NP-6AK agonist was more effective than the control for up regulation of MCL-1.

FIGS. 3A and 3B show that an NP-6AK agonist promotes neurite elongation (FIG. 3A) and reverses rapamycin-mediated suppression of MCL-1 expression (FIG. 3B) in SH-SY5Y cells.

FIG. 4 shows data from studies with ex vivo tissue that demonstrated that an NP-6AK agonist provided neuroprotection to normal primary murine embryonic cortical neurons in culture when challenged with nutrient deprivation. For nutrient deprivation, primary cortical neurons (14 DIV) were incubated in glucose-free Locke's medium with or without 300 nm NP-6AK agonist. Cells treated with an NP-6AK agonist had a 60% increased activity (p<0.05) compared with negative controls, corroborating previous data showing increased cell viability under conditions of serum starvation and neuroprotection.

DETAILED DESCRIPTION

AT2R activation is suppressed in a variety of disease states including hypertension, diabetes, cancers and various neurodegenerative diseases. Suppression of AT2R leads to increased activity of the AT1R which is a major contributor to metabolic diseases (e.g., cardiovascular and renal diseases, type 2 diabetes) and cancers. Therefore, the present invention provides a new class of laboratory reagents and therapeutic polypeptides which can be used to characterize and treat such disorders.

References and Definitions

The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued U.S. patents, pending U.S. applications, published foreign patents and applications, and references, including protein and nucleotide database sequences, that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

As used herein, the terms “about” or “approximately” mean within twenty percent (20%) of the numerical amount cited.

As used herein, the term “a” means one or more.

As used herein, the terms “increased” or “decreased” mean at least 10% more or less, respectively, relative to pre-treatment with an agonist of the invention.

As used herein, a “pharmaceutical composition” includes an active agent and a pharmaceutically acceptable carrier.

As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce a severe allergic, pyrogenic or similarly undesired reaction when administered to a mammal.

As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or other aqueous solutions, saline solutions, aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin (61).

Polypeptide Modulators of AT2R Activity

The invention provides a new class of agonists which are polypeptides (or polypeptide derivatives or analogues) comprising 6 amino acid residues with the generic sequence A1-A2-A3-A4-A5-A6, where A1 is Lys; A2 is Pro, 3Hyp or 4Hyp; A3 is Leu or Ile; A4 is Lys, A5 is Pro, 3Hyp or 4Hyp, and A6 is Trp (SEQ ID NO. 1). This new class is referred to as NP-6AK agonists. Sequences with Hyp in at least one position may be preferred due to increased stability. Other derivatives or analogues of these agonists may include chemical modifications that increase stability in the bloodstream for use as a pharmaceutical reagent.

One possible modification is formation of non-natural peptide bonds for additional stability or the attachment of the side chain atoms to a different atom of the residue. An example of such chemistry is cited in Hook et al. (60). The authors describe beta amino acids, wherein the side chains are attached to the beta carbon, whereas natural amino acid side chains are attached to the alpha carbon. Various studies have shown that these “beta peptides” are less likely to be degraded by non-specific peptidases compared to natural peptides. Any such chemistry that modifies the natural peptide for additional stability could be used, including peptoids in which the side chain is attached to the nitrogen.

Another method of stabilizing the polypeptides of the invention is covalent or non-covalent association with an inert water-soluble polymer. When administered systemically, therapeutic compositions are often cleared rapidly from the circulation and may therefore elicit relatively short-lived pharmacological activity. Consequently, frequent injections of relatively large doses of bioactive compounds may be required to sustain therapeutic efficacy. Any water-soluble (e.g., at least about 0.01 mg/ml) inert polymer which provides the conjugate with the desired increase in stability or half-life is suitable for use in the invention. Non-proteinaceous polymers are particularly preferred. The polymer is preferably a hydrophilic synthetic polymer, such as a polyvinyl polymer (e.g., polyvinylalcohol and polyvinylpyrrolidone), polyalkylene ether (e.g., polyethylene glycol (PEG)); polyoxyalkylene (e.g., polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics)); polymethacrylate; or carbomer. However, natural polymers are also useful, such as branched or unbranched polysaccharides which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g., polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid including, for example, lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrins, glycogen, or polymers of sugar alcohols such as polysorbitol and polymannitol, heparin or heparan. The molecular weight of the polymer can range from about 10,000 to 500,000 Daltons (D), and may typically be about 20,000 D, about 30,000 D, about 40,000 D, or about 50,000 D.

Compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol (PEG), copolymers of polyethylene glycol and polypropylene glycol, or monomethoxypolyethylene glycol (mPEG), carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds. Such modifications may also increase the composition's solubility in aqueous solution, reduce aggregation, increase the physical or chemical stability of the compound, and reduce the immunogenicity and reactivity of the composition.

Attachment of polyethylene glycol (PEG) to agonist compositions of the invention is particularly useful because PEG has very low toxicity in mammals and may reduce the immunogenicity or antigenicity of the agonist compositions. Numerous activated forms of PEG suitable for direct reaction with proteins have been described. Useful PEG reagents for reaction with protein amino groups include active esters of carboxylic acid or carbonate derivatives, particularly those in which the leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or 1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing maleimido or haloacetyl groups are useful reagents for the modification of protein free sulfhydryl groups. Likewise, PEG reagents containing amino hydrazine or hydrazide groups are useful for reaction with aldehydes generated by periodate oxidation of carbohydrate groups in proteins.

The agonist compositions of the present invention may be delivered in a microencapsulation device so as to reduce or prevent a host immune response against the polypeptide or against cells which may produce the polypeptide. The polypeptide or compositions of the present invention may also be delivered microencapsulated in a membrane, such as a liposome. As an example, polymers such as PEG may be conveniently attached to one or more reactive amino acid residues in a polypeptide of the agonist compositions, such as the alpha-amino group of the amino terminal amino acid, the epsilon amino groups of lysine side chains, the sulfhydryl groups of cysteine side chains, the carboxyl groups of aspartyl and glutamyl side chains, the alpha-carboxyl group of the carboxy-terminal amino acid, tyrosine side chains, or to activated derivatives of glycosyl chains attached to certain asparagine, serine or threonine residues.

Another method of modifying the polypeptide agonists of the invention is to add a signal sequence to the N- or C-terminus. The term “signal sequence,” as used herein, refers to any short peptide that directs the trafficking of a protein in the cell. Signal sequences may, for example, direct secretion of a polypeptide, or localization within an intracellular compartment. Signal sequences also frequently determine the orientation of a peptide across a cell membrane. One example is an N-terminal sequence of about 20 amino acids that directs secretory and transmembrane proteins to the endoplasmic reticulum (ER) (see, e.g., von Heijne (1985), J. Mol. Biol. 184:99-105). Signal sequences may also be engineered to include one or more specific protease recognition sites, such that the signal sequences will be removed by endogenous proteases after trafficking.

AT2R Agonist Reagents

Activation of AT2R in laboratory testing is of high interest due to the many effects of AT2R described above. Therefore, in one aspect, the invention provides AT2R agonists as in vitro or in vivo reagents for laboratory research.

Thus an NP-6AK agonist to AT2R has utility as a control to characterize and quantify downstream effects of the receptor activation, such as its effects on the mammalian target of rapamycin (MTOR), NHE6, ErbB3 and Nitric Oxide Synthase.

For example, CHO cells expressing AT2R and MTOR are treated with an NP-6AK agonist and insulin to activate both MTOR and AT2R. AT2R suppresses MTOR-mediated phosphorylation of Ribosomal Protein S6 (RPS6). Western blotting can be used to determine RPS6 phosphorylation state which is decreased by at least 10% in response to AT2R activation by the agonist NP-6AK. The same cell line can then be treated with a different AT2R agonist candidate to assess the efficacy of the agonist candidate, or the same cell line can be treated with both an AT2R agonist of the invention and an AT2R antagonist candidate (e.g., EMA300, Smith et al. (2013), Pain Medicine 14(10):1557-68; PD123319, Chakrabarty et al. (2008), Endocrinology 149(7):3452) to assess the efficacy and mode of action (i.e., competitive, non-competitive) of the antagonist candidate.

An NP-6AK agonist increases expression levels of at least one, and in some embodiments all three, MCL-1 isoforms.

Methods of Treatment

In order to treat an AT1R-mediated inflammatory response or an inflammatory response arising from under-activation of the AT2R, and/or the symptoms arising therefrom, an NP-6AK agonist is administered by any route that will permit delivery of the active agent to the affected cells. In some embodiments, administration is subcutaneous, intramuscular or intraperitoneal, but may also be by inhalation, intra-arterial, intravenous, intradermal, topical, oral, parenteral, intraventricular, or intracranial administration. Alternatively, the active agent may be delivered locally to the system or the affected cells by any suitable means.

In therapeutic treatments of the invention, a therapeutically effective amount of the pharmaceutical composition is administered to a mammalian patient. As used herein, the term “therapeutically effective amount” means an amount sufficient to reduce by at least 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably substantially eliminate or prevent, a clinically significant metric or deficit in the activity, function and response of the patient. Specifically, a therapeutically effective amount will cause one or more of the following: decreased AT1R activation, increased AT2R activation; decreased cortisol levels; stabilized insulin levels; decreased pro-inflammatory cytokines, decreased pro-inflammatory interleukins, increased function of dopaminergic neurons, decreased Reactive Oxygen Species, decreased mucous production, or a decrease or increase in any other relevant markers as discussed herein or that would be known to one of ordinary skill in the art as it relates to cystic fibrosis (CF), diabetes, metabolic X syndrome; hyperglycemia, autism, Alzheimer's disease, inflammation or cancer. The frequency and dosage of the therapy can be titrated by the ordinary physician or veterinarian using standard dose-to-response techniques that are well known in the art.

Pharmaceutical Formulations

Liquid forms, such as lotions suitable for topical administration or for cosmetic application, may include a suitable aqueous or non-aqueous vehicle with buffers, suspending and dispensing agents, thickeners, penetration enhancers, and the like. Solid forms such as creams or pastes or the like may include, for example, any of the following ingredients, water, oil, alcohol or grease as a substrate with surfactant, polymers such as polyethylene glycol, thickeners, solids and the like. Liquid or solid formulations may include enhanced delivery technologies such as liposomes, microsomes, microsponges and the like.

The above-described components for liquid, semisolid and solid topical compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pa., which is incorporated herein by reference.

When pharmaceutical compositions are to be administered transdermally they typically are employed as liquid solutions or as gels. In these settings the concentration of agonists of the present invention range from about 0.1% to about 20%, and preferably from about 0.1% to about 10%, of the composition with the remainder being aqueous mixed or non-aqueous vehicle, such as alcohols and the like, suspending agents, gelling agents, surfactant, and the like. Examples of suitable such materials are described below.

The agonist-containing compositions of this invention can also be administered in sustained release transdermal forms or from transdermal sustained release drug delivery systems. A description of representative sustained release materials can be found in the incorporated materials in Remington's Pharmaceutical Sciences, supra.

The agonist compositions for systemic administration include compositions for oral administration, that is liquids and solids, and compositions for injection.

Compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical solvent. Typical unit dosage forms include profiled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. According to one embodiment, an agonist composition of the present invention is usually a minor component (from about 0.01 to about 20% by weight or preferably from about 0.1 to about 15% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.

Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature including a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an solvent such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

According to another embodiment, injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. A compound of the present invention in such compositions is typically a minor component, about 0.1-30% by weight, with the remainder being the injectable carrier and the like.

The above-described components for orally administrable or injectable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in the part of Remington's Pharmaceutical Sciences noted above.

EXAMPLES

The NP-6AK agonists if the invention, including polypeptides comprising, consisting of, or consisting essentially of the sequences Lys-Pro-Leu-Lys-Pro-Trp (SEQ ID NO. 2); Lys-3Hyp-Leu-Lys-Pro-Trp (SEQ ID NO. 3); Lys-4Hyp-Leu-Lys-Pro-Trp (SEQ ID NO. 4); Lys-Pro-Ile-Lys-Pro-Trp (SEQ ID NO. 5); Lys-3Hyp-Ile-Lys-Pro-Trp (SEQ ID NO. 6); Lys-4Hyp-Ile-Lys-Pro-Trp (SEQ ID NO. 7); Lys-Pro-Leu-Lys-3Hyp-Trp (SEQ ID NO. 8); Lys-3Hyp-Leu-Lys-3Hyp-Trp (SEQ ID NO. 9); Lys-4Hyp-Leu-Lys-3Hyp-Trp (SEQ ID NO. 10); Lys-Pro-Ile-Lys-3Hyp-Trp (SEQ ID NO. 11); Lys-3Hyp-Ile-Lys-3Hyp-Trp (SEQ ID NO. 12); Lys-4Hyp-Ile-Lys-3Hyp-Trp (SEQ ID NO. 13); Lys-Pro-Leu-Lys-4Hyp-Trp (SEQ ID NO. 14); Lys-3Hyp-Leu-Lys-4Hyp-Trp (SEQ ID NO. 15); Lys-4Hyp-Leu-Lys-4Hyp-Trp (SEQ ID NO. 16); Lys-Pro-Ile-Lys-4Hyp-Trp (SEQ ID NO. 17); Lys-3Hyp-Ile-Lys-4Hyp-Trp (SEQ ID NO. 18); and Lys-4Hyp-Ile-Lys-4Hyp-Trp (SEQ ID NO. 19), can be tested in various cell-based systems to demonstrate AT2R agonist activity, and utility in the methods described herein.

Thus, an NP-6AK agonist comprising the sequence Lys-Pro-Leu-Lys-Pro-Trp (SEQ ID NO. 2) promoted cell survival across mouse HL-1 cardiomyocytes and human smooth vascular muscle cells by acting through AT2R activation. Cell survival was assessed by measurement of cell index (CI) using a Xcelligence™ Real-Time Cell Analyzer (Westburg B V, Leusden, N L) to quantitatively determine the effect of different treatments on cell survival, adhesion, and proliferation. Cell lines treated with the NP-6AK agonist (300 nM) demonstrated significantly better survival and more proliferation than those treated with CGP42112A (300 nM) (Sigma-Aldrich, Co. LLC, St. Louis, Mo.), a partial agonist of AT2R. CGP42112A and the NP-6AK agonist (300 nM) increased CI of serum starved HL-1 cells in the order CGP42112A (≥14%), NP-6AK (≥25%). Increase in CI is a direct effect of AT2R activation. This was verified by addition of an AT2R antagonist, PD123319 (Sigma-Aldrich, Co. LLC, St. Louis, Mo.). In the presence of this antagonist, the cell survival otherwise observed using the NP-6AK agonist was not present.

An NP-6AK agonist upregulated MCL-1 by selectively activating AT2R across several cell lines. This effect was observed in cardiomyocytes, human vascular smooth muscle cells, SH-SY5Y cells (ATCC, Manassas, Va.), and PC-12 neuronal cells (ATCC, Manassas, Va.) in conditions of serum starvation and/or toxicity. When incubated with an NP-6AK agonist at a concentration of 300 nM, cardiomyocytes displayed 45% higher MCL-1 expression and human smooth vascular muscle cells displayed 22% higher MCL-1 expression (FIG. 1). When any of these cell lines was pre-treated with an AT2R antagonist such as PD123319, the MCL-1 upregulation was not observed, implying that NP-6AK activation of AT2R is necessary for MCL-1 upregulation. Cells treated with the NP-6AK agonist generated higher MCL-1 upregulation relative to those treated with a partial agonist such as CGP42112A.

The SH-SY5Y cell line is a neuronal cell line with both dopaminergic and adrenergic receptors. When this cell line was treated with Rapamycin, MCL-1 expression was reduced. Addition of an NP-6AK agonist to this cell line after treatment with Rapamycin was able to recover MCL-1 expression. When just the NP-6AK agonist was added at a concentration of 300 nM, these cells displayed 2-fold increase in MCL-1 expression. See FIG. 2.

Serum-starvation reduced survival of SH-SY5Y cells and PC-12 cells. When these cell lines were treated with an NP-6AK agonist, they had a higher survival rate and demonstrated increased MCL-1 expression and neurite elongation. See FIG. 3. When PC-12 cells were treated with AT2R antagonist PD123319, the protective effects of the NP-6AK agonist were lost, indicating that the NP-6AK agonist's direct action is through AT2R activation. Treatment of this cell line with 300 nM of NP-6AK agonist led to increased cell viability by over 30% relative to treatment with native ligand of AT2R Ang II (300 nM). Relative to treatment with 300 nM CGP42112A, cells treated with an NP-6AK agonist displayed over 70% increased survival. Treatment with AngII or CGP42112A was more detrimental relative to results obtained when cells are treated with AT2R antagonist PD123319. AT2R activation by NP-6AK agonist promotes cell survival whereas AT2R activation by other ligands are harmful to it. These results were assessed using an MTS cell proliferation assay (Biovision Inc., Milpitas, Calif.).

Ex vivo neurons were obtained and treated with an NP-6AK agonist in different conditions to assess neuroprotective properties. Normal primary murine embryonic cortical neurons in culture (14 days in vitro) were challenged with nutrient deprivation by incubation in glucose-free Locke's medium (Schnapf et al. (1990)). These cultures were then supplemented with 300 nM NP-6AK agonist or no NP-6AK agonist. Cultures that were supplemented 300 nm NP-6AK agonist demonstrated 60% increased activity (p<0.05) compared with negative controls. See FIG. 4.

Zucker Obese (ZO) rats (Charles River Laboratories, Inc., Wilmington, Mass.), a diabetic animal model displaying signs of cardiovascular disease were treated for 2 weeks (dose of 0.9 mg/kg/day) via subcutaneous injection with an NP-6AK agonist and assessed for several markers of cardiovascular health including blood markers and structural parameters of the heart. Controls were ZO rats that received saline. Fasting plasma and urine profiles demonstrated that animals receiving the NP-6AK agonist had reduced triglycerides (˜50%), urine protein (˜68%), reduced urine N-acetyl-beta-A-glucosaminidase (˜60%), and increased HDL by 12% on average. Echocardiography performed using the Vevo® 2100 platform (VisualSonics, Toronto, Ontario, CA) indicated that animals treated with the NP-6AK agonist had improved structural cardiac parameters including circumferential strain of endocardium (short axis view), and myocardial performance index (MPI) (p≤0.005), and E/E′ ratio (p≤0.002), a powerful predictor of primary cardiac events. See Table 1.

TABLE 1 Treatment groups Plasma Profile Urine Profile (2 Weeks; Saline or mg/dL mg/dL Echocardiography/Strain analysis NP-6A 0.9 mg/kg/day) Triglycerides HDL Protein NA6 MPI E/E Circumferential Strain Male ZO rat ± Saline 1229 ± 164 65 ± 4.1 1426.7 ± 649.5 27.9 ± 6.4 0.516 ± 0.03 32.3 ± 2.06 −20.56 ± 1.65 Male ZO rat ± NP-6A  610 ± 109 73 ± 2.8   445 ± 86.4 10.9 ± 1.5 0.389 ± 0.02  26 ± 2.1 −26.11 ± 2.47

Although the invention has been described in detail with particular reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited herein are hereby incorporated by reference.

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What is claimed:
 1. A pharmaceutical composition comprising a polypeptide comprising the amino acid sequence that corresponds to the formula SEQ ID NO: 1 A1-A2-A3-A4-A5-A6 wherein: A1 is Lys; A2 is Pro, 3Hyp or 4Hyp; A3 is Leu or Ile; A4 is Lys; A5 is Pro, 3Hyp or 4Hyp; and A6 is Trp.
 2. A pharmaceutical composition comprising a polypeptide consisting essentially of the amino acid sequence that corresponds to the formula SEQ ID NO: 1 A1-A2-A3-A4-A5-A6 wherein: A1 is Lys; A2 is Pro, 3Hyp or 4Hyp; A3 is Leu or Ile; A4 is Lys; A5 is Pro, 3Hyp or 4Hyp; and A6 is Trp.
 3. A pharmaceutical composition comprising a polypeptide consisting of the amino acid sequence that corresponds to the formula SEQ ID NO: 1 A1-A2-A3-A4-A5-A6 wherein: A1 is Lys; A2 is Pro, 3Hyp or 4Hyp; A3 is Leu or Ile; A4 is Lys; A5 is Pro, 3Hyp or 4Hyp; and A6 is Trp.
 4. The pharmaceutical composition of claim 1 wherein the polypeptide has Angiotensin II Type 2 Receptor (AT2R) agonist activity when administered to a cell expressing an AT2R receptor.
 5. The pharmaceutical composition of claim 1 wherein: when A2 is Pro, A5 is 3Hyp or 4Hyp; or when A5 is Pro, A2 is 3Hyp or 4Hyp.
 6. The pharmaceutical composition of claim 1 wherein the composition is lyophilized.
 7. The pharmaceutical composition of claim 1 further comprising a pharmaceutically acceptable carrier. 